Potential sources of metabolic chemical energy that thermophiles can use in a hot spring ecosystem can be quantified by combining analytical and thermodynamic data, as in this case for Obsidian Pool at Yellowstone National Park. This hot spring is chosen because of extensive previous efforts to characterize its microbial community structure with molecular methods, as well as successful efforts to isolate microorganisms from the pool and study their physiology. We quantify the potential metabolic energy available from 182 reactions that are out of equilibrium in Obsidian Pool, rank the sources by energy availability, and categorize the resulting energy structure by electron acceptor. The results provide the first comprehensive view of the distribution of energy supplies from chemical reactions in a hot spring ecosystem. Hypotheses derived from these results can be tested through experiments on the relative rates of proposed metabolic processes, genomic and proteomic analyses of natural samples, and informed efforts to isolate and study novel organisms.